Reconstructive surgery is presently performed without real-time, objective intraoperative assessment. As such, reconstructive surgeons face three major problems in decision-making: flap design, assessment of flap thrombosis, and treatment of flap salvage. Important decisions regarding each of these are now based solely on subjective criteria rather than patient-specific objective data. Because intraoperative decisions ultimately lead to the success or failure of a flap, our study focuses on the application of recent advances in optical imaging technology to provide the reconstructive surgeon with objective assessment of flap design, flap viability, and candidacy for flap salvage. Our laboratory has previously developed a near-infrared (NIR) fluorescence imaging system for intraoperative use and has validated its utility in sentinel lymph node mapping and other surgeries. This system uses harmless, invisible NIR light, makes no contact with the patient, and has no moving parts. Surgical anatomy is displayed in color simultaneously with an overlay of NIR fluorescence to assess, objectively, specific areas of interest. Our laboratory also has considerable experience with the chemistry of NIR fluorophores and has developed several targeted NIR fluorescence contrast agents. In this application, we describe pre-clinical and clinical studies whose long-range goal is to provide the plastic and reconstructive surgeon with real-time, objective image guidance during complex surgeries. In Specific Aim 1, we develop sensitive, real-time, intraoperative techniques for patient-specific flap planning using an NIR fluorophore that is already FDA-approved. In Specific Aim 2, we assess tissue and flap thrombosis before, during, and after reconstructive procedures using long-circulating autologous NIR fluorescent platelets developed by our laboratory. In Specific Aim 3, we guide decision-making regarding salvage therapy with fibrinolytics using an NIR fibrinogen derivative also developed by our laboratory. Finally, in Specific Aim 4, we translate the results from our pre-clinical studies to human reconstructive surgery during a Phase I clinical trial utilizing an FDA-approved NIR fluorophore. With the completion of these studies, we foresee being able to solve major clinical problems in reconstructive surgery using real-time, intraoperative objective assessment.